QC Report


general
Report generated at2025-02-21 23:41:53
TitlePancreas
DescriptionATAC-seq data from Liu et al. pancreas data processed in 2025
Pipeline versionv2.2.0
Pipeline typeatac
Genomemm10
Alignerbowtie2
Sequencing endedness{'rep1': {'paired_end': True}, 'rep2': {'paired_end': True}}
Peak callermacs2

Alignment quality metrics


SAMstat (raw unfiltered BAM)

rep1rep2
Total Reads35834434107188668
Total Reads (QC-failed)00
Duplicate Reads00
Duplicate Reads (QC-failed)00
Mapped Reads3334671799968988
Mapped Reads (QC-failed)00
% Mapped Reads93.1000000000000193.30000000000001
Paired Reads35834434107188668
Paired Reads (QC-failed)00
Read11791721753594334
Read1 (QC-failed)00
Read21791721753594334
Read2 (QC-failed)00
Properly Paired Reads3042554492017284
Properly Paired Reads (QC-failed)00
% Properly Paired Reads84.8999999999999985.8
With itself3190631495763600
With itself (QC-failed)00
Singletons14404034205388
Singletons (QC-failed)00
% Singleton4.03.9
Diff. Chroms7715674573
Diff. Chroms (QC-failed)00

Marking duplicates (filtered BAM)

rep1rep2
Unpaired Reads00
Paired Reads1206009636323994
Unmapped Reads00
Unpaired Duplicate Reads00
Paired Duplicate Reads6540792445759
Paired Optical Duplicate Reads00
% Duplicate Reads5.42356.7332

Filtered with samtools flag 1804 (samtools view -F 1804):


Fraction of mitochondrial reads (unfiltered BAM)

rep1rep2
Rn = Number of Non-mitochondrial Reads3319862899465529
Rm = Number of Mitochondrial Reads184786618828
Rm/(Rn+Rm) = Frac. of mitochondrial reads0.0055352637090981760.006183064152572814

SAMstat (filtered/deduped BAM)

rep1rep2
Total Reads2276027667614240
Total Reads (QC-failed)00
Duplicate Reads00
Duplicate Reads (QC-failed)00
Mapped Reads2276027667614240
Mapped Reads (QC-failed)00
% Mapped Reads100.0100.0
Paired Reads2276027667614240
Paired Reads (QC-failed)00
Read11138013833807120
Read1 (QC-failed)00
Read21138013833807120
Read2 (QC-failed)00
Properly Paired Reads2276027667614240
Properly Paired Reads (QC-failed)00
% Properly Paired Reads100.0100.0
With itself2276027667614240
With itself (QC-failed)00
Singletons00
Singletons (QC-failed)00
% Singleton0.00.0
Diff. Chroms00
Diff. Chroms (QC-failed)00

Filtered and duplicates are removed. Subsampling with atac.subsample_reads is not done in alignment steps. Nodup BAM is converted into a BED type (TAGALIGN) later and then TAGALIGN is subsampled with such parameter in the peak-calling step.

Fragment length statistics (filtered/deduped BAM)

rep1rep2
Fraction of reads in NFR0.71157828628802410.7100783510400204
Fraction of reads in NFR (QC pass)TrueTrue
Fraction of reads in NFR (QC reason)OKOK
NFR / mono-nuc reads2.6903919273314642.6385425585045663
NFR / mono-nuc reads (QC pass)TrueTrue
NFR / mono-nuc reads (QC reason)OKOK
Presence of NFR peakTrueTrue
Presence of Mono-Nuc peakTrueTrue
Presence of Di-Nuc peakFalseFalse

rep1
rep1
rep2
rep2

Open chromatin assays show distinct fragment length enrichments, as the cut sites are only in open chromatin and not in nucleosomes. As such, peaks representing different n-nucleosomal (ex mono-nucleosomal, di-nucleosomal) fragment lengths will arise. Good libraries will show these peaks in a fragment length distribution and will show specific peak ratios.



Sequence quality metrics (filtered/deduped BAM)

rep1
rep1
rep2
rep2

Open chromatin assays are known to have significant GC bias. Please take this into consideration as necessary.


Annotated genomic region enrichment

rep1rep2
Fraction of Reads in universal DHS regions0.59689829771835810.578667008606471
Fraction of Reads in blacklist regions0.0060214999150273930.003990283703551205
Fraction of Reads in promoter regions0.17983947119094690.17362450572542115
Fraction of Reads in enhancer regions0.4127232024778610.4024473394953489

Signal to noise can be assessed by considering whether reads are falling into known open regions (such as DHS regions) or not. A high fraction of reads should fall into the universal (across cell type) DHS set. A small fraction should fall into the blacklist regions. A high set (though not all) should fall into the promoter regions. A high set (though not all) should fall into the enhancer regions. The promoter regions should not take up all reads, as it is known that there is a bias for promoters in open chromatin assays.


Library complexity quality metrics


Library complexity (filtered non-mito BAM)

rep1rep2
Total Fragments1202351136196537
Distinct Fragments1139015933867478
Positions with Two Read4916051671907
NRF = Distinct/Total0.9473240.935655
PBC1 = OneRead/Distinct0.953170.944815
PBC2 = OneRead/TwoRead22.08432219.138931

Mitochondrial reads are filtered out by default. The non-redundant fraction (NRF) is the fraction of non-redundant mapped reads in a dataset; it is the ratio between the number of positions in the genome that uniquely mapped reads map to and the total number of uniquely mappable reads. The NRF should be > 0.8. The PBC1 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with AT LEAST one read pair. PBC1 is the primary measure, and the PBC1 should be close to 1. Provisionally 0-0.5 is severe bottlenecking, 0.5-0.8 is moderate bottlenecking, 0.8-0.9 is mild bottlenecking, and 0.9-1.0 is no bottlenecking. The PBC2 is the ratio of genomic locations with EXACTLY one read pair over the genomic locations with EXACTLY two read pairs. The PBC2 should be significantly greater than 1.


Fragment: read for a single-ended dataset, pair of reads for a paired-ended dataset
NRF: non redundant fraction
PBC1: PCR Bottleneck coefficient 1
PBC2: PCR Bottleneck coefficient 2
PBC1 is the primary measure. Provisionally


Replication quality metrics


IDR (Irreproducible Discovery Rate) plots

rep1_vs_rep2
rep1_vs_rep2
rep1-pr1_vs_rep1-pr2
rep1-pr1_vs_rep1-pr2
rep2-pr1_vs_rep2-pr2
rep2-pr1_vs_rep2-pr2
pooled-pr1_vs_pooled-pr2
pooled-pr1_vs_pooled-pr2

Reproducibility QC and peak detection statistics

overlapidr
Nt12883975160
N18548841164
N212465071960
Np13647381579
N optimal13647381579
N conservative12883975160
Optimal Setpooled-pr1_vs_pooled-pr2pooled-pr1_vs_pooled-pr2
Conservative Setrep1_vs_rep2rep1_vs_rep2
Rescue Ratio1.05925224505002371.0854044704630121
Self Consistency Ratio1.45809938236945551.74812943348557
Reproducibility Testpasspass

Reproducibility QC


Number of raw peaks

rep1rep2
Number of peaks153507199099

The number of peaks is capped at 300000
Peaks are called from macs2 with p-val threshold 0.01

Peak calling statistics


Peak region size

rep1rep2idr_optoverlap_opt
Min size150.0150.0150.0150.0
25 percentile191.0209.0486.0343.0
50 percentile (median)286.0332.0745.0538.0
75 percentile520.0626.01094.0876.0
Max size2444.03082.03087.03087.0
Mean408.490765893412479.91451991220447824.6770124664436659.4317484044464

rep1
rep1
rep2
rep2
idr_opt
idr_opt
overlap_opt
overlap_opt

Enrichment / Signal-to-noise ratio


TSS enrichment (filtered/deduped BAM)

rep1rep2
TSS enrichment12.86887916857924812.728467987530006

rep1
rep1
rep2
rep2

Open chromatin assays should show enrichment in open chromatin sites, such as TSS's. An average TSS enrichment in human (hg19) is above 6. A strong TSS enrichment is above 10. For other references please see https://www.encodeproject.org/atac-seq/


Jensen-Shannon distance (filtered/deduped BAM)

rep1rep2
AUC0.121610903746174730.16077010805100822
Synthetic AUC0.489963897873149650.49418751744417083
X-intercept0.390462783740981870.17221617903476077
Synthetic X-intercept2.758692794537362e-846.740138456396787e-254
Elbow Point0.74849231359877140.7641490569961448
Synthetic Elbow Point0.499222694262407730.5055456531927898
Synthetic JS Distance0.4568810030968860.4749334025884419

Peak enrichment


Fraction of reads in peaks (FRiP)

FRiP for macs2 raw peaks

rep1rep2rep1-pr1rep2-pr1rep1-pr2rep2-pr2pooledpooled-pr1pooled-pr2
Fraction of Reads in Peaks0.32350666573639090.32218140734851120.313606741851460870.307473248238832540.3127793353648260.308048511674463840.33376520655446720.3153630831063040.3151767473919307

FRiP for overlap peaks

rep1_vs_rep2rep1-pr1_vs_rep1-pr2rep2-pr1_vs_rep2-pr2pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks0.294601965005267640.272873931757242340.28860000496936740.3018841063558227

FRiP for IDR peaks

rep1_vs_rep2rep1-pr1_vs_rep1-pr2rep2-pr1_vs_rep2-pr2pooled-pr1_vs_pooled-pr2
Fraction of Reads in Peaks0.246494421060025360.199885449543757730.24132243444576170.25640433858589073

For macs2 raw peaks:


For overlap/IDR peaks: